Goggatomy: A Method for Opening Small Cuticular Compartments in Arthropods for Physiological Experiments.

Department of Cellular and Molecular Physiology, Yale University New Haven, CT, USA.

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Department of Chemical Engineering, University of Iowa Iowa, IA, USA.

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Department of Dentistry, University of Iowa Iowa, IA, USA.

Abstract

Most sense organs of arthropods are ensconced in small exoskeletal compartments that hinder direct access to plasma membranes. We have developed a method for exposing live sensory and supporting cells in such structures. The technique uses a viscous light cured resin to embed and support the structure, which is then sliced with a sharp blade. We term the procedure a "goggatomy," from the Khoisan word for a bug, gogga. To demonstrate the utility of the method we show that it can be used to expose the auditory chordotonal organs in the second antennal segment and the olfactory receptor neurons in the third antennal segment of Drosophila melanogaster, preserving the transduction machinery. The procedure can also be used on other small arthropods, like mosquitoes and mites to expose a variety of cells.

Schematic of the goggatomy procedure. (A) Sequence of steps illustrated for a Drosophila head (read left to right). The gray rectangle represents the surface of a plastic petri dish. (B) Approximate level of section to produce intact JOs. (C) Goggatomized head in chip of LCR inserted into wax. (D) Higher powered view of a different sectioned head. Scale bar 100μm.

Light microscopic images of JOs within a goggatomized A2. (A) Bright field image of JO (B) fluorescent image of JO expressing GCaMP6. Both specimens have approximately the same orientation. Scale bars 20μm.

Free-Arista Goggatomy. (A) Schematic of the procedure (1) Place the cut head on a small droplet of LCR (blue) and allowed it to sink so that its edges, but not the antennae or arista, are covered. Cure for 20 s. (2) Place a small strip of colored Cellophane (gray rectangle) over the A3, to prevent LCR from pulling the antennae up when it is applied to A2. (3) Apply a very small droplet of LCR so that it covers the dorsal margins of the A2 (green). Begin light curing, then remove the plastic strip and apply a droplet of water to the preparation. Cure for 1 min. Wick off the water with a tissue. (4) Apply DS and cut through the head as indicated by the dotted line. (B) Response of JO cells expressing GCaMP6 in a FA-goggatomized preparation to an air pulse (120 ms, ~ 1 psi) directed at the solution (red arrow). Inset top, fluorescence, bottom pseudo-color difference image. Scale bar 20μm.

Sectioning A3 and response of ORNs to odorant. (A) Schematic of the procedure. Place the cut head on a droplet of LCR (blue) and allow sinking so that the margins of the head, but not the arista, become covered. Light cure for 20 s. Apply small droplets of LCR over the arista and margins of the antennae (green). Note, the frontal surface of A3 should not be covered so that the sensory sensilla are free. Add droplet of water over preparation and cure for 1 min. Wick off water with tissue. Add droplet of DS and cut through the A3s (red line). (B) Image of goggatomize A3 expressing GCaMP6. (C) Increase in intracellular calcium in ORNs expressing GCaMP6 to the application of iso-amyl acetate (arrow, a 5 μl drop of a 0.67 mM solution was added to the ~2 ml bath). Upper inset, fluorescence prior to odorant application. Lower inset pseudo-color difference image. Scale bar 20μm.

Mounting live flies with LCR. (A) Schematic of the mounting procedure. (B) Movement of arista (inset right) induced by a single sound pulse (red). (C) Response of JO expressing ArcLight in the same fly as (B). Scale bar 20μm.